Nurr1 is a transcription factor that structurally resembles members of the superfamily of nuclear hormone receptors. DNA binding domains of these receptors contain two zinc fingers and usually a ligand and dimerization domain confined to the COOH-terminus. Since a ligand for Nurr1 has not been identified, it is also considered an orphan receptor. Nurr1 expression appears to be predominantly brain specific detected in embryonic stages with the peak expression at birth or soon thereafter. Most striking is its expression in the substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain, which contains most of the brain dopamine/DA/cell bodies. To ascertain the function of this orphan receptor, we generated Nurr1-null mice by homologous recombination. At birth, Nurr1-null mutant mice were indistinguishable from their Nurr1 wild type and heterozygous +/- littermates. Within a few hours, however, after the first feeding, the Nurr1-null mice were readily distinguishable by absence of milk in the stomach and by a curved posture. Within 12 hours after birth, they appeared to be weak, became dehydrated, and died. In contrast, Nurr1 +/- mutants thrived, reproduced, and displayed overt phenotype. Attempts to rescue Nurr1-null pups by decreasing the size of litters or using NIH Swiss foster mothers were unsuccessful. Histological and morphological analyses of newborn Nurr1-null pups showed no defects in any major organ. Based on Nurr1 expression in dopaminergic neurons, we assayed for catechol contents in brains of wild type and mutant mice by high-performance liquid chromatography. In Nurr1-null mice the concentration of DA was decreased by 90%, whereas the level of norepinephrine was unchanged when compared with the wild type. Using immunohistochemistry and/or in situ hybridization we showed that absence of Nurr1 prevents the appearance of tyrosine hydroxylase, the rate of limiting enzyme in DA synthesis, aromatic amino and decarboxylase, vesicular monoamine transporter and DA membrane transporter selectively in the nigrostrial and mesolimbic-mesocortical systems, regions affected in Parkinson's disease. The results point to DA deficiency from a failure to develop the dopaminergic phenotype. Using another set of markers, we observed no changes in cell density or in organization in the SN and VTA. Furthermore, the neuron-specific marker confirmed the presence of neurons in these regions. Taken together, we provided evidence for an additional mechanism to account for DA depletion in vivo, in addition to disruption of the tyrosine hydroxylase gene or dopaminergic neurotoxins treatment. The mechanism(s) whereby the Nurr1 orphan nuclear receptor governs this process, however, is unclear. Basis for a role of Nurr1 in acquisition of the DA neuronal phenotype in the ventral midbrain and the nature of the neurons in the SN and VTA of Nurr1-null mice are being investigated. Presently, it is not known what factors are responsible for Nurr1 selective expression. TTo understand the mechanism controlling Nurr1 expression, we have created transgenic mice containing approximately 3 kilobases (kb) of Nurr1 gene 5' flanking region driving the expression of the reporter gene LacZ. This construct has been established in the germ line of two separate mice. The progeny of these mice will be analyzed by staining brain sections for LacZ expression, which should determine if the 3 kb Nurr1 genomic fragment is sufficient for tissue and cell-type specific expression. The promoter region also has been analyzed by primer extension. Several RNA transcription start sites have been identified. In conjunction with the primer extension experiments the Nurr1 promoter has been analyzed in transfection experiments. PC12 cells were transfected with various fragments of the Nurr1 promoter fused to the luciferase reporter gene. These results revealed that the strongest transcription activating regions of Nurr1 are close to exon 1, and that expression decreases when Nurr1 promoter sequences distal to exon 1 are added. These results indicate that the distal promoter fragments may contain sites for transcriptional repressors.